Jia Qiong, Ren Hongrui, Zhang Shuyin, Yang Haoyu, Gao Shuaipeng, Fan Ruiwen
College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China.
Vet Sci. 2024 Aug 19;11(8):381. doi: 10.3390/vetsci11080381.
All subtypes of () produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by . In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti- toxin-related therapeutic products.
（）的所有亚型都会产生α毒素（CPA），该毒素可导致羔羊、牛、猪和马患肠炎或肠毒血症，以及人类和动物患创伤性梭菌性肌坏死。CPA作用于细胞膜，最终导致内吞作用和细胞死亡。因此，中和CPA对于预防和治疗由（）引起的疾病至关重要。在本研究中，以CPA作为抗原，从羊驼的天然纳米抗体库中制备了一种半衰期为2.9小时、亲和常数（KD）为0.9 nmol/L且在60℃以下具有良好稳定性的纳米抗体（CPA-VHH）。CPA-VHH的生物学活性分析表明，它能够以15倍的比例有效中和CPA的磷脂酶和溶血活性。在Vero细胞中，9.8 μg/mL的CPA-VHH在半数最大抑制浓度（IC）的两倍时中和了CPA的细胞毒性。在小鼠模型中，35.7 ng/g体重（BW）的CPA-VHH中和了由2×半数致死剂量（LD）的CPA所导致的90%的致死率。研究发现，在2×LD CPA的情况下，CPA-VHH在30分钟内保护了80%的小鼠，但2小时后降至50%以下，4小时后降至0%。救援试验表明，在感染2×LD CPA后30分钟内使用CPA-VHH，救援率达到80%，2小时后降至10%。此外，CPA-VHH有效减轻了紧密连接蛋白-1（ZO-1）、闭合蛋白和Claudin-1表达水平的降低，同时也减弱了由CPA感染诱导的促炎细胞因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-7（IL-7）、白细胞介素-8（IL-8）、肿瘤坏死因子α（TNF-α）和干扰素-γ（IFN-γ）的上调。总体而言，本研究鉴定出了一种特异性纳米抗体CPA-VHH，它在体外和动物模型中均能有效中和CPA毒素，为抑制这些毒素的致病性提供了一种新工具，并为开发新的抗毒素相关治疗产品奠定了重要基础。
